Detection of analytes e.g., proteins, nucleic acids, DNA, bacterial fragments or other compounds, is important for both molecular biology research and medical applications. For example, detection of analytes allows determining the effect of an experimental treatment or the effect of a DNA mutation over all the biomolecules in a sample.
Several detection methods of analytes are available.
Enzyme-linked immunosorbent assay (ELISA) is used to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample. It involves immobilizing an antigen to a solid surface and complexing the antigen with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The reading of the measurable product is usually performed by chemiluminescence whose optical emission is weak.
Other techniques based on the detection of fluorescent molecules are widely used. These techniques comprise grafting a fluorescent molecule to the target analyte and analyzing the fluorescent emission. However, this approach requires many steps to isolate the grafted molecule of interest, and is limited by the weakness of the fluorescence signal and the photo degradation of the graft.
To avoid these issues, Gold Nano Particles (GNP) have attracted attention for bioassay development. GNPs can be prepared in a broad range of diameters (2 to 250 nm) with a high degree of precision and accuracy. Once prepared, they are stable for long periods, and because they are generally employed at very low concentrations they are economic to use even though the material from which they are made is relatively expensive. They are easily functionalized with recognition molecules (antibodies, antigens, oligonucleotides, etc.), thereby leading to highly stable conjugates. Assays using GNPs are mostly based on their optical and catalytic properties of GNPs. Metal nanoparticles have unique optical properties arising from their ability to support a localized surface plasmon resonance. More precisely, the localized surface plasmon resonance is the collective oscillation of the nanostructure conduction band electrons in resonance with the incident electromagnetic field. The spectrum of the localized surface plasmon resonance is strongly reliant upon the nanostructure size, shape and composition and the dielectric constant of the surrounding environment. As a result, a solution of nanoparticles has a characteristic color which can change depending on changes in the nanoparticles themselves and/or in the arrangement of the nanoparticles. It is these unique properties which have led to the development of metal nanoparticle based sensor technologies.
For example, a selective colorimetric detection method using GNP probes was developed by Elghanian et al (see “Selective Colorimetric Detection of Polynucleotides based on the distance-dependent Optical Properties of Gold Nanoparticles” Science Vol. 277 (1997)). Hybridization of the probe comprising GNPs with the target forms aggregates, causing a color visual change of the gold colloid solution from red (absence of hybridization) to purple (hybridization).
In order to obtain information on the particle dynamics with accuracy and chemical specificity, Jans et al, in “Dynamic light scattering as a powerful tool for gold nanoparticle bioconjugation and biomolecular binding studies” Anal. Chem. 81, 9425-9432 (2009), reports a technique which couples the use of Gold Nano Particle (GNP) probe as a light-scattering enhancer and Dynamic Light Scattering (DLS) as a read out system. The basic principle of DLS is as follows: a sample is illuminated by a laser beam and scattered light fluctuations due to the Brownian motion of the particle are detected at a known scattering angle by a fast photon detector. DLS instruments that measure the scattered light fluctuation as function of time and at a fixed angle can determine the mean particle size in a limited size range.
WO2009117168 also relates to an analytical method for the detection of an analyte using metal nanoparticles as light-scattering enhancers and DLS as a read out system. In this technique, a metal nanoparticle is conjugated with a plurality of identical or different receptors to form probes; a sample solution is formed by mixing the solution expected to include the analytes and the probes. In case of the presence of analytes, said analytes will bind to the receptors of the probes. To detect the presence of analytes, a light is directed towards the sample solution and the fluctuation over time of the light scattered by the sample solution is measured. From this measurement, DLS is used to quantify the amount of analyte by measuring the degree of aggregation in the solution. Aggregates comprising the analytes are distinguished from isolated probes in analyzing the nature of the DLS signal. However, this method shows a low signal to noise ratio which results in a limited sensitivity and a low specificity.
By sensitivity, it is meant the capacity to detect a minimum number of analytes per unit of volume. By specificity, it is meant the capacity to distinguish a specific analyte with a maximum of confidence.
The present invention proposes an original method and system for detecting the presence of an analyte, using nanoparticles as light scattering enhancers, which provides an excellent signal to noise ration and a very good specificity.